Guidelines for Setting Up PCR Reactions (NEB #E6631)

The NEBNext Direct protocol incorporates Illumina adaptor sequences; therefore, the libraries generated from this protocol may be sequenced on Illumina platforms including the MiSeq, NextSeq®, and HiSeq platforms. Here we describe the steps necessary to sequence NEBNext Direct libraries on the Illumina MiSeq.

If samples are run on the Illumina NextSeq, please note that while the i5 index is generated in the reverse complement orientation, no adjustments need to be made because the i5 UMI is a random sequence.

4.1.1. Reconfigure the MiSeq Reporter software to write both index reads to FASTQs.

By default, the Illumina MiSeq is set to not generate FASTQs for index reads. It is necessary to change this setting in order to make use of the 8 base sample barcode in the i7 position (index read 1) and the 12 base UMI in the i5 position (index read 2).

4.1.1.1. Open the MiSeq Reporter configuration file (C:\Illumina\ MiSeq Reporter\MiSeqReporter.exe.config).

4.1.1.2. Edit the “CreateFastqForIndexReads” setting in the MiSeq Reporter configuration file by adding the following line (or edit the existing line so the value is “1”) between the <appSettings> tags:

<add key=”CreateFastqForIndexReads” value=”1”/>

4.1.1.3 Save the updated file on the MiSeq as C:\Illumina\MiSeq Reporter\MiSeqReporter.exe.config

4.1.2. Prepare a MiSeq sample sheet:

4.1.2.1. Download a MiSeq sample sheet from the “Other Tools & Resources” tab on the NEBNext Direct website at: https:// www.neb.com/E6631 – NEBNext Direct Custom Ready Panels (NEB #E6631). Do not use the Illumina Experiment Manager to generate a sample sheet.

4.1.2.2. Fill in the empty fields with your sample and barcode information.

4.1.2.3 Transfer the sample sheet file (*.csv) to the Miseq and save the file in D:\Illumina\Miseq Control Software\SampleSheets\38

4.1.3. Pool, dilute, and denature samples* for an 8 pM** final concentration following the MiSeq Denature and Dilute Libraries Guide and the Index Pooling Guidelines .

* The number of samples that can be pooled together will depend on the input amount, panel size, and number of reads required for each sample for the particular analysis being performed. For guidelines on the number of reads required for a desired target coverage, see Figure 1.2A & 1.2B, page 27–28.

** Based on quantification by a QubitdsDNA HS Assay Kit, Agilent® High Sensitivity DNA Kit, or Agilent High Sensitivity D1000 ScreenTape. For other quantification methods, this concentration may need to be empirically determined for optimal cluster density.

4.1.4. Follow the MiSeq System Guide to load your samples and run the MiSeq. When prompted, upload the sample sheet that was prepared in 4.1.2.